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Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates to and from model membranes by the LE/LY phospholipid lysobisphosphatidic acid (LBPA). It had been previously shown that enrichment of NPC1-deficient cells with LBPA results in cholesterol clearance. Here we demonstrate that LBPA enrichment in human NPC2-deficient cells, either directly or via its biosynthetic precursor phosphtidylglycerol (PG), is entirely ineffective, indicating an obligate functional interaction between NPC2 and LBPA in cholesterol trafficking. We further demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Together these studies reveal a heretofore unknown step of intracellular cholesterol trafficking which is critically dependent upon the interaction of LBPA with functional NPC2 protein. Cholesterol is a type of fat that is essential for many processes in the body, such as repairing damaged cells and producing certain hormones. Normally, cholesterol enters cells from the bloodstream and is then moved to the parts of the cell that need it via a process known as ‘trafficking’. When cholesterol trafficking goes wrong, abnormally large amounts of cholesterol and other fats accumulate within the cell. Over time, these fatty deposits become toxic to cells and eventually damage the affected tissues. Niemann-Pick type C disease (NPC) is a severe genetic disorder affecting cholesterol trafficking. It is characterized by cholesterol build-up in multiple tissues, including the brain, which ultimately causes degeneration and death of nerve cells. Two proteins, NPC1 and NPC2, are involved in NPC disease. Both proteins normally help move cholesterol out of important trafficking compartments (known as the endosomal and lysosomal compartments) to other areas of the cell where it is needed. Patients with the disease can have mutations in either the gene for NPC1 or the gene for NPC2. This means that cells from NPC1 patients do not make enough functional NPC1 protein (but contain working NPC2), and vice versa. Previous studies had shown that giving cells with NPC1 mutations large amounts of the small molecule lysobisphosphatidic acid (LBPA for short) could compensate for the loss of NPC1, and stop the toxic build-up of cholesterol. McCauliff, Langan, Li et al. therefore wanted to explore exactly how LBPA was doing this. They had shown that LBPA dramatically increased the ability of purified NPC2 protein to transport cholesterol, and wondered if the effect of LBPA in the cells without NPC1 depended on NPC2. They predicted that boosting LBPA levels would not work in cells lacking NPC2. Biochemical experiments using purified protein showed that LBPA and NPC2 did indeed interact directly with each other. Systematically changing different building blocks of NPC2 revealed that a single region of the protein is sensitive to LBPA, and when this region was altered, LBPA could no longer interact with NPC2. Since LBPA is naturally produced by cells, they then stimulated cells grown in the laboratory to generate more LBPA using its precursor phosphatidylglycerol. They used cells from patients with mutations in either NPC1 or NPC2 and demonstrated that LBPA’s ability to reverse the accumulation of cholesterol was dependent on its interaction with NPC2. Thus, increasing LBPA levels in cells from patients with NPC1 mutations was beneficial, but had no effect on cells from patients with NPC2 mutations. These results shed new light not only on how cells transport cholesterol, but also on potential methods to combat disorders of cellular cholesterol trafficking. In the future, LBPA could be developed as a genetically tailored, patient-specific therapy for diseases like NPC.

Background Small RNAs regulate gene expression in species across the tree of life. miRNAs, which impact a variety of cellular and physiological processes ranging from development and stress adaptation to host defense, are one of the best characterized classes of small RNA. Many miRNAs are produced from longer non-coding transcripts generated from host genes via a series of RNA cleavage reactions. The location of a small RNA within a host gene can shape the processing of the mature small RNA. For example, a type of miRNAs derived from host gene intronic sequence, referred to as miRtrons, are Drosha-independent and reliant on splicing for biogenesis. Relatedly, processing of a small RNA from an exon of a protein-coding mRNA, in principle, may destabilize it and compromise translation of the host gene. Prior to extensive transcriptome analysis, informatics analyses identified six human miRNAs embedded in exons of protein-coding genes and experimental studies have characterized additional anecdotal examples. Still, whether protein-coding mRNAs encoding small RNAs represent an appreciable class of host genes given the now recognized complexity of the transcriptome is unclear. Results Our analysis finds 201 small RNAs (118 human and 83 mouse) encoded by expressed exons of protein-coding genes (5’-UTR, CDS, 3’-UTR). Forty-six of these cases (29 human and 17 mouse) are also present in MirGeneDB which includes the most up-to-date miRNA classifications. Many of these small RNAs are poorly characterized with 96% of the protein-coding host gene relationships identified here not previously known. Furthermore, the identification of nearly fifty human and mouse small RNAs embedded within coding exons of canonical ORFs suggests that overlapping hybrid genes might be more common than previously appreciated in higher organisms. Expression analysis for a subset of these small RNAs indicates that many display differential expression across human tissues with the pattern correlating significantly with the expression of the candidate protein-coding host gene. Significance Overall, our analysis suggests that the number of protein-coding transcripts serving as host genes is greater than previously recognized. Our small RNA host gene classifications may serve as a resource to shed new light on small RNA biology, specific host genes, and gene regulation.

Purpose of ReviewA significant fraction of allergens bind small molecular ligands, and many of these compounds are classified as lipids. However, in most cases, we do not know the role that is played by the ligands in the allergic sensitization or allergic effector phases.Recent FindingsMore effort is dedicated toward identification of allergens’ ligands. This resulted in identification of some lipidic compounds that can play active immunomodulatory roles or impact allergens’ molecular and allergic properties.SummaryFour allergen families (lipocalins, NPC2, nsLTP, and PR-10) are among the best characterized in terms of their ligand-binding properties. Allergens from these four families are able to bind many chemically diverse molecules. These molecules can directly interact with human immune system and/or affect conformation and stability of allergens. While there is more data on the allergens and their small molecular ligands, we are just starting to understand their role in allergy.

Olfactory binding proteins are essential components of the highly sensitive olfactory system in insects. They play crucial roles in detecting, binding, and transporting environmental odorants and pheromones to olfactory receptors. Although a large number of olfactory binding proteins have been identified in insects to date, research in this field continues to advance rapidly. This review summarizes recent progresses in understanding their structures, functions, mechanisms of action, and potential applications. Structurally, these proteins typically form simple, stable, spherical conformations composed of α-helices and/or β-sheets, which support environmental adaptability and diverse physiological functions. Two main hypotheses have been proposed to explain their mechanisms of action: pH-dependent regulation and ligand-induced conformational changes. In terms of practical applications, olfactory binding proteins have shown great promise in biological pest control, the breeding of economically important insects, and the development of biosensors, making them attractive targets for future research and innovation.

Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease.

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor in the world. Sorafenib is the first-line drug for patients with advanced HCC. However, long-term treatment with sorafenib often results in reduced sensitivity of tumor cells to the drug, leading to acquired resistance. Identifying biomarkers which can predict the response to sorafenib treatment may represent a clinical challenge in the personalized treatment era. Niemann-Pick type C2 (NPC2), a secretory glycoprotein, plays an important role in regulating intracellular free cholesterol homeostasis. In HCC patients, downregulation of hepatic NPC2 is correlated with poor clinical pathological features through regulating mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) activation. This study aimed to investigate the roles of secretory NPC2-mediated free cholesterol levels as biomarkers when undergoing sorafenib treatment and evaluate its impact on acquired sorafenib resistance in HCC cells. Herein, we showed that NPC2 downregulation and free cholesterol accumulation weakened sorafenib’s efficacy through enhancing MAPK/AKT signaling in HCC cells. Meanwhile, NPC2 overexpression slightly enhanced the sorafenib-induced cytotoxic effect. Compared to normal diet feeding, mice fed a high-cholesterol diet had much higher tumor growth rates, whereas treatment with the free cholesterol-lowering agent, hydroxypropyl-β-cyclodextrin, enhanced sorafenib’s tumor-inhibiting ability. In addition, sorafenib treatment induced higher NPC2 secretion, which was mediated by inhibition of the Ras/Raf/MAPK kinase (MEK)/ERK signaling pathway in HCC cells. In both acquired sorafenib-resistant cell and xenograft models, NPC2 and free cholesterol secretion were increased in culture supernatant and serum samples. In conclusion, NPC2-mediated free cholesterol secretion may represent a candidate biomarker for the likelihood of HCC cells developing resistance to sorafenib.

Background: The Niemann–Pick C2 (NPC2) gene family plays an important role in olfactory communication, immune response, and the recognition of host plants associated with environmental adaptation for insects. Methods: In this study, we conducted a genomic analysis of the structural characteristics and physicochemical properties of the NPC2 genes of eleven butterfly species available, focusing on the alpine Parnassius species, especially Parnassius glacialis, to investigate their duplication and expression patterns. Results: Our results indicate that a significant expansion of NPC2 genes was detected in P. glacialis compared to other butterflies; in addition, the expansion of these unevenly distributed P. glacialis NPC2 chromosome genes was shaped by tandem duplication mediated by transposons. Furthermore, the PgNPC2 genes had relatively higher expression in P. glacialis antennae and other head tissues. These facts were verified by quantitative real-time PCR (qRT-PCR). Conclusions: These findings suggest that the expansion of NPC2 genes may have contributed to the local adaptation of P. glacialis during its dispersal ‘out of the Qinghai–Tibet Plateau’, although further functional tests are needed to confirm their specific role in this adaptive process.

Niemann-Pick disease type C (NPC), is a rare lysosomal storage disorder, which has a variable presentation based on the age of onset. We describe five adult/adolescent-onset NPC cases presenting with a range of movement disorders along with vertical supranuclear gaze palsy as part of the clinical presentation. A diagnostic delay of 4–17 years from the symptom onset was found in this case series. A high index of clinical suspicion in adult/adolescent patients presenting with vertical supranuclear gaze palsy along with various movement disorder phenomenology can help in the early diagnosis of NPC.

A handoff model has been proposed to explain the egress from lysosomes of cholesterol derived from receptor-mediated endocytosis of LDL. Cholesterol is first bound by soluble Niemann-Pick C2 (NPC2) protein, which hands off the cholesterol to the N-terminal domain of membrane-bound NPC1. Cells lacking NPC1 or NPC2 accumulate LDL-derived cholesterol in lysosomes and fail to deliver LDL cholesterol to the endoplasmic reticulum (ER) for esterification by acyl-CoA acyltransferase (ACAT) and for inhibition of sterol regulatory element-binding protein cleavage. Here, we support this model by showing that the cholesterol transport defect in NPC1 mutant cells is restricted to lysosomal export. Other cholesterol transport pathways appear normal, including the movement of cholesterol from the plasma membrane to the ER after treatment of cells with 25-hydroxycholesterol or sphingomyelinase. The NPC1 or NPC2 block in cholesterol delivery to the ER can be overcome by 2-hydroxypropyl-β-cyclodextrin, which leads to a marked increase in ACAT-mediated cholesterol esterification. The buildup of cholesteryl esters in the cytosol is expected to be much less toxic than the buildup of free cholesterol in the lysosomes of patients with mutations in NPC1 or NPC2.

Background Niemann-Pick disease type C (NP-C) is a rare neurovisceral lipid storage disorder characterised by progressive, disabling neurological symptoms and premature death in most patients. During the last decade, national cohort studies have accrued a great deal of data on the symptomatology and natural history of NP-C. Methods In an observational cohort study, we present a substantial update based on the clinical presentation and follow-up of all known UK-based patients with a confirmed diagnosis of NP-C who have been tracked on an electronic database at the Department of Genetic Medicine, University of Manchester, UK. Patients were stratified according to accepted age-at-neurological-onset categories. Data on patients’ clinical signs and symptoms, medical history and genetic studies are summarised using descriptive methods. Results A total of 146 patients with NP-C were included, representing the full known UK NP-C cohort, as observed from database information between 1999 and the end of 2011: 72 patients (49 %) were alive at the end of the observation period. Among a total of 116 patients (79 %) who possessed at least one identified, disease-causing NP-C gene mutation, 114 (98 %) had NPC1 and two (2 %) had NPC2 mutations. Overall, 53/194 (27 %) identified mutations were novel. Six patients (4 %) had an early, non-neurological neonatal onset form of NP-C. The numbers (%) of patients with accepted age-at-neurological onset forms were: 8 (5 %) early-infantile onset, 51 (35 %) late-infantile onset, 42 (29 %) juvenile onset, and 25 (17 %) adolescent/adult onset. Fourteen patients diagnosed based on visceral symptoms and/or sibling history, confirmed in most cases by genetic analysis, did not have any neurological manifestations at last follow up (11 patients with mean [SD] age at last follow up 2.5 [1.8] years: 3 with mean [SD] age at death 20.8 [15.9] years). A total of 51 patients (35 %) received miglustat therapy. The mean (SD) overall treatment duration up to the end of the observation period was 2.6 (2.3) years. Conclusions This UK cohort is the largest national NP-C cohort reported to date, and confirms the wide phenotypic variability of the disease, as reported in other countries. Further analyses are required to assess the impact of miglustat therapy on neurological disease progression.